Genetic transformation is a methodology used in the field of life sciences and as such used in various organisms for many purposes. This technology has had and has major implications in many areas of life sciences research. One aspect of the technology is that it can be used to identify functions of individual nucleotide molecules (e.g. genetic elements and/or genes) or proteins encoded by such nucleotide molecules. The resulting genetically modified organism can be used in fundamental or applied research or be used in an industrial application.
Genetic transformation can be a powerful technology in the field of plant sciences as it allows the transfer of a nucleotide molecule of interest to a receiving plant species. Such a nucleotide molecule can comprise promoters, genes, terminators, repressors or enhancers of gene of protein function, etcetera.
Genetic transformation can for example be used with the intention to study the effects of addition of a protein of interest to a receiving plant. In such cases, a gene that codes for the protein of interest, which is usually part of a larger nucleotide molecule, is introduced in the receiving plant.
In order to obtain a genetically modified plant, a method can be applied which comprises transformation followed by regeneration and subsequent development of transformed, regenerated plant material into a mature transgenic plant. Such a method can be successfully applied only to particular plant species that are responsive to both the transformation and regeneration phase and subsequent further development into a mature transgenic plant. Such plant species include for example Arabidopsis thaliana or Brassica napes. 
During transformation a nucleotide molecule of interest is introduced into a plant cell. During regeneration, transformed plant material is allowed to develop from rather undefined structures, such as callus tissue, into plant organs, such as leaf like-structures, shoot-like structures or somatic embryos, such that a mature transgenic plant can be obtained therefrom.
The transformation phase can comprise contacting of a plant cell or plant material with an Agrobacterium tumefaciens bacterium which contains a Ti (Tumour-inducing) plasmid having the nucleotide molecule of interest. The Ti plasmid comprises at least a DNA segment which is transferred by Agrobacterium tumefaciens to a host-plant; the T-DNA element. The T-DNA element is flanked by DNA repeats, the so called left border and right border. Genetic engineering allows one to place a nucleotide molecule of interest between the left and right T-DNA border of the Ti plasmid. During contact of the plant cell or plant material with the Agrobacterium tumefaciens bacterium, at least the T-DNA element of the Ti plasmid, including the nucleotide molecule of interest, is transferred from the Agrobacterium tumefaciens bacterium into the plant cell where it is stably integrated in the nuclear genome or organellar genome, such as from a mitochondrion or chloroplast. Other methods of transformation may also be applied to plant material.
When considering suitable regeneration from a macroscopic point of view, regenerating plant cells or tissues may develop into an amorphous mass of cells (i.e. callus) from which a shoot-like structure or leaf-like structure can develop. Usually from such structures an elongated stem can develop, if needed under the influence of suitable plant hormones. Subsequently, such structures will, if needed under the influence of one or more suitable root-inducing agents, initiate formation of a root system to develop an advanced root system suitable to sustain further development. Subsequently, plant material of a suitable advanced stage of developmental can be transplanted from an in-vitro to an ex-vitro environment. The plant is subsequently grown ex-vitro, such as on soil, vermiculite, rock wool or the like, under suitable conditions which allow obtaining a mature transgenic plant. The regeneration procedure may comprise additional or alternative steps of this general concept. Alternatively, regeneration may proceed through the formation of somatic embryos which may be allowed to grow into mature plants.
Such a method of transformation and regeneration is preferably applied to young somatic plant tissues, cultured cells such as protoplasts or organs as starting material. Such tissues, such as explants of young plant tissue or pieces of plant material from seedlings, comprise cells of variable degrees of differentiation or determination. Likely due to the heterogeneous population of cells of various levels or degrees of differentiation which are present in such tissues, are such tissues in particular responsive to the initial phase of a regeneration treatment.
Only certain plants or plant species have been shown to be responsive to transformation and regeneration methods. It has been possible to apply existing methods to transform and regenerate such plants in a relatively straightforward manner. Conversely, it has become evident that other plants or plant species are unresponsive to such transformation and regeneration methods. Such plants do not regenerate in a suitable manner and/or cannot be made to regenerate at all into mature plants. Such plants, plant varieties or plant cultivars are called “recalcitrant” or “regeneration incompetent”.
It is largely unknown which underlying molecular or physiological factors are responsible or that determine whether plants are recalcitrant or not. This indicates the current need for developing reliable plant-specific transformation and regeneration methods which can be applied to a wide variety of plant species. In fact, suitable transformation and regeneration methods for the efficient provision of mature transgenic plants have only been developed for a few species or cultivars of species.
A plethora of different problems has been observed and found to be insurmountable when recalcitrant plants were subjected to transformation and regeneration methods followed by development into mature transgenic plants. Such problems comprise inability to transform a plant cell from subjected plant cells or plant material. Or in case transformation can be achieved, such transformed material may not develop into a mature transformed plant. Transformed cells or plant material may regenerate up until a certain developmental stage, display aberrant behaviour such as altered or aberrant growth, premature termination of development, severely delayed development, or incorrect development. Also, the formation of false-positive plants is known to occur. Such non-transformed plant material may escape from the pressure of a selective agent and regenerate into a mature plant.
Furthermore, the following problems with respect to the applicability, reliability and suitability of the method are known: reproducibility can be problematic; the outcome of the method can be unpredictable, the amount of regenerating shoots, roots or plantlets may be too low for suitable application in an industrial setting; the method can only applied to a single plant species, or to a particular group of cultivars of a given plant species; the method may not be suitable for high-throughput or routine application; the method may depend on a specific bacterial strain for suitable applicability. It may be clear that such methods are not suitable for cost-efficient industrial applications. Hence, there remains a need for efficient and reliable methods for the provision of mature transgenic plants which can be applied to recalcitrant plant species.